This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term goal of the proposed research is to understand how genomic integrity is preserved in cells. Highly complex surveillance mechanisms, consisting of DMA repair, DMA replication and checkpoint proteins, have developed to maintain genomic integrity. Dysfunction of these mechanisms can lead to a variety of clinical symptoms including an increased risk of cancer. Progress in our understanding of the downstream effectors proteins activated in response to DMA damage that lead to cell cycle arrest and DNA repair have been made but the mechanisms by which DNA damage is detected and signaled remain elusive. Therefore, we aim to define the sensor components of the replication stress response pathway and determine how they work cooperatively in stabilizing stalled replication forks during S-phase of the cell cycle. Among the proteins thought to be involved in this response include the MRN complex (composed of MRE11, RAD50 and NBS1), and RPA, (RPA, the major single-stranded DNA binding protein in eukaryotic cells composed of three subunits p70, p34, p14), proteins that are intricately involved in DNA metabolism and maintenance of genomic stability. We have recently identified a protein/protein interaction between the MRN complex and RPA. We believe that the MRN complex and RPA work together in response to DNA damage and stalled replication forks. However, how the MRN complex and RPA sense and initiate a response to stalled replication forks remains undefined. This proposal will test the hypothesis that stalled replication forks stimulate the MRN complex in an RPA-dependent manner to stabilize and repair damage at all sites of stalled replication. To test this hypothesis the following aims are proposed: Aim 1: To characterize the direct protein-protein interactions of RPA and the MRN complex. Aim 2: Define the role of RPA in the recruitment of the M/R/N complex to sites of stalled replication. Aim 3: Characterize the role of RPA and phosphorylation in the tethering of DNA by the MRN complex.